This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding sterol metabolism enzymes in plants and seeds.
Conversion of 7-dehydrocholesterol to cholesterol is the last reaction in the cholesterol biosynthesis pathway catalyzed by the microsomal enzyme 7-dehydrocholesterol-delta 7 reductase (EC 1.3.1.21). Inhibiting the last step in cholesterol biosynthesis profoundly reduces tissue and plasma cholesterol concentrations and accumulates precursors that substantially slow hepatoma growth. Inhibiting late cholesterol synthesis also hinders the growth of rapidly enlarging malignant tumors (Xu, G. et al. (1996) Hepatology 24:440-445). Analyses of the cDNA encoding the human delta 7 sterol reductase shows that this enzyme is a membrane-bound protein containing 6 to 9 putative transmembrane segments and is structurally related to plant and yeast sterol reductases. The delta 7 sterol reductase is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human delta 7 reductase cDNA remove the C7-8 double bond in 7-dehydrocholesterol in a NADPH-dependent manner (Moebius, F. F. et at. (1998) Proc. Natl. Acad. Sci. USA 95:1899-1902).
A microsomal preparation from seedlings of Zea mays catalyzed the NADPH-dependent reduction of the delta 7 bond of delta 5,7 cholestadienol giving the first in vitro evidence of the intermediacy of delta 5,7 sterols in plant sterol biosynthesis. In vitro inhibition of the plant delta 5,7 sterol delta 7 reductase by ammonium ion-containing fungicides was consistent with the previously proposed cationic mechanism involved in this reduction reaction (Taton, M. and Rahier, A. (1991) Biochem. Biophys. Res. Commun. 181:465-473). The NADPH-sterol delta-7 reductase from Arabidopsis thaliana has been cloned. The corresponding protein has significant sequence similarity with yeast delta 14 and delta 24 reductases and with human lamin B receptor. This protein is capable of efficiently reducing in vivo delta-5,7-ergosta- and cholesta-sterols, regardless of the structural variations on the side chain. The delta 7 reductase activity is preferentially associated with the endoplasmic reticulum membrane and uses NADPH as the reducing agent (Lecain, E. et al. (1996) J. Biol. Chem. 271:10866-10873).
Regulation of sterol biosynthesis in the terminal portion of the pathway represents an efficient mechanism by which the cell can control the production of sterol without disturbing the production of other essential mevalonate pathway products. Expression of ERG3, the gene encoding sterol C-5 desaturase, is increased in response to a mutation in the major isoform of HMG-CoA reductase which catalyzes the rate-limiting step of sterol biosynthesis. Mutations in non-auxotropic ergosterol biosynthetic genes downstream of squalene production result in an up-regulation of ERG3 expression. Absence of sterol esterification leads to a decrease in total intracellular sterol and ERG3 is a target of this negative regulation (Arthington-Skaggs, B. A. et al. (1996) FEBS Lett. 392:161-165). ERG3 is the structural gene in Saccharomyces cerevisiae for the sterol delta 5 desaturase that introduces the C5=6 unsaturation in ergosterol biosynthesis. Inactivated mutants of ERG3 fail to grow without added levels of delta 5 sterols in heme-deficient cells, and are unable to grow on the respiratory substrates glycerol and ethanol (Smith, S. J. and Parks, L. W. (1993) Yeast 9:1177-1187). A construct containing the promoter for the ERG3 gene fused to the bacterial lacZ reporter gene was placed in strains making aberrant sterols, and the effect of altered sterol composition on gene expression was monitored by beta-galactosidase activity. The absence of ergosterol resulted in a 35-fold increase in the expression of ERG3 as measured by beta-galactosidase activity. The level of ERG3 mRNA was increased as much as 9-fold in erg-mutant strains or wild-type strains inhibited in ergosterol biosynthesis by antifungal agents. The observed regulatory effects of ergosterol on ERG3 are specific for ergosterol, as several ergosterol derivatives failed to elicit the same controlling effect. These results demonstrate that ergosterol exerts a regulatory effect on gene transcription in S. cerevisiae (Smith, S. J. et al. (1996) Mol. Cell Biol. 16:5427-5432). A human cDNA clone homologous to fungal ERG3, a gene encoding sterol C-5 desaturase has been isolated. This gene is expressed in all normal human tissues examined (Matusushima, M. et al. (1996) Cytogent. Cell Genet. 74:252-254).
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 30 amino acids that has at least 84% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn sterol delta-7 reductase polypeptide of SEQ ID NO:2, a rice sterol delta-7 reductase polypeptide of SEQ ID NO:4, a soybean sterol delta-7 reductase polypeptide of SEQ ID NO:6, a wheat sterol delta-7 reductase polypeptide of SEQ ID NO:8, a rice sterol delta-7 reductase of SEQ ID NO:18, a soybean sterol delta-7 reductase of SEQ ID NO:20, and a wheat sterol delta-7 reductase of SEQ ID NO:22, or a corn sterol-C5 desaturase polypeptide of SEQ ID NO:10, a rice sterol-C5 desaturase polypeptide of SEQ ID NO:12, a soybean sterol-C5 desaturase polypeptide of SEQ ID NO:14, a wheat sterol-C5 desaturase polypeptide of SEQ ID NO:16, a corn sterol-C5 desaturase of SEQ ID NO:24, a rice sterol C-5 desaturase of SEQ ID NO:26, a soybean sterol-C5 desaturase of SEQ ID NO:28, and a wheat sterol C-5 desaturase of SEQ ID NO:30. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 30 amino acids that has at least 84% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotides of the claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 40 (preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29 and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a sterol delta-7 reductase or a sterol-C5 desaturase polypeptide of at least 30 amino acids comprising at least 84% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a sterol delta-7 reductase or a sterol-C5 desaturase polypeptide in a host cell, preferably a plant cell, the method comprising the steps of:
constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene into a host cell;
measuring the level a sterol delta-7 reductase or a sterol-C5 desaturase polypeptide in the host cell containing the isolated polynucleotide; and
comparing the level of a sterol delta-7 reductase or a sterol-C5 desaturase polypeptide in the host cell containing the isolated polynucleotide with the level of a sterol delta-7 reductase or a sterol-C5 desaturase polypeptide in a plant cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a sterol delta-7 reductase or a sterol-C5 desaturase polypeptide gene, preferably a plant sterol delta-7 reductase or a sterol-C5 desaturase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 40 (preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a sterol delta-7 reductase or a sterol-C5 desaturase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a sterol delta-7 reductase or a sterol-C5 desaturase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a sterol delta-7 reductase or a sterol-C5 desaturase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a sterol delta-7 reductase or a sterol-C5 desaturase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of sterol delta-7 reductase or a sterol-C5 desaturase in the transformed host cell; (c) optionally purifying the sterol delta-7 reductase or a sterol-C5 desaturase expressed by the transformed host cell; (d) treating the sterol delta-7 reductase or a sterol-C5 desaturase with a compound to be tested; and (e) comparing the activity of the sterol delta-7 reductase or a sterol-C5 desaturase that has been treated with a test compound to the activity of an untreated sterol delta-7 reductase or a sterol-C5 desaturase, thereby selecting compounds with potential for inhibitory activity.
The present invention relates to a composition comprising the isolated polynucleotide of the present invention.
The present invention relates to a composition comprising a polypeptide of the present invention.
The present invention relates to an expression cassette comprising an isolated polynucleotide of the present invention operably linked to a promoter.
The present invention relates to a method for positive selection of a transformed cell comprising:
(a) transforming a plant cell, preferably a monocot or a dicot, with a chimeric gene of the present invention or an expression cassette of the present invention; and
(b) growing the transformed plant under conditions allowing expression of the polynucleotide in an amount sufficient to modify the amount of sterol in the cell to provide a positive selection means.
The present invention relates to the method of the present invention, wherein the plant cell is a monocot.
The present invention relates to the method of the present invention, wherein the plant cell is a dicot.
As used herein, the following terms shall apply:
xe2x80x9cSterol metabolism enzymesxe2x80x9d refers to sterol delta-7 reductase and/or sterol-C5 desaturase.